The present invention relates to the production of plant cell lines by microspore or anther culture, and to a hybridization method that entails the use of plants regenerated from such cell lines ("regenerants"), thereby to exploit sporophytically controlled self-incompatibility.
Three biological methods have been employed for the production of various hybrids: cytoplasmic male sterility, genetic male sterility, and self-incompatability. Other chemical and mechanical methods have also been used. Cytoplasmic male sterility has been used to produce hybrids of oilseed rape (Brassica napus), while self-incompatibility is the method commonly used to produce hybrids of the vegetable Brassica crops like Brussel sprouts, cauliflower, broccoli and cabbage.
There are two types of self-incompatibility in plants, namely, sporophytic and gametophytic. The present invention relates to sporophytically-determined self-incompatibility, a condition under which pollen does not readily germinate and fertilize if it lands on the stigma of its own flowers or of the flowers of other plants carrying the same allele. The stigma of a self-incompatible plant is receptive, however, to pollen from a plant that does not contain the same self-incompatibility (SI) determinant, even though the pollen-donor plant may contain another SI determinant.
The prevention of fertilization in an incompatible pollination is due to the action of two mechanisms at the stigmatic surface. There is poor adhesion of the pollen grains to the surface and pollen tube growth is inhibited or reduced so that fertilization fails to take place. S. Gowers in PROC. EUCARPIA "CRUCIFERAE 1979" CONF. (Wageninger) 80-84 (hereafter "Proceedings").
In this description, the term "determinant" denotes a unit character of Mendelian heredity. Although some characters, particularly of a quantitative nature, are due to a number of genes, self-incompatibility is generally controlled by a single gene which may be dominant or recessive. In Brassica, for example, self-incompatibility is controlled by two genes, each represented by two alleles, at two different loci.
More specifically, self-incompatibility has been recognized in B. napus, see Olsson (1953) K. Landtbr. Akad. Tidskr. 92: 394-402, as well as in B. campestris, see Bateman (1955) Heredity (London) 9: 53-68, and in B. cleracea, see Thompson (1957) J. Genet. 55: 45-60. The use of self-incompatibility to produce a double-cross hybrid of marrow stem kale has been suggested by Thompson (1959) Agriculture 65: 487-91. The use of self-incompatibility as a means of producing oilseed rape was also proposed by Gowers, (1975), Euphytica 24: 537-41, Proceedings 80-84, (1980) Eucarpia cruciferae Newslet. 5: 15-16, who outlined a system for producing modified, double-cross F1 hybrid seed using normal and self-incompatible isogenic lines.
The maintenance of SI lines has been a major problem. There are at least three methods which can be used to overcome the self-incompatibility and, thus, to reproduce such lines: micropropagation, variation of the levels of carbon dioxide and other chemical treatments, and bud pollination. Micropropagation is a new procedure, presently being explored, that involves the reproduction of a plant from single cells or clumps of cells derived from suitable tissue. The use of carbon dioxide is one of the preferred methods. It involves the raising of carbon dioxide levels around the plant within a few hours after the pollen has landed on the stigma. While this method can be used in a greenhouse, it cannot be used on a field scale. In bud pollination, each individual flower must be dissected before it has fully developed and opened, and pollen then deposited on the immature stigma, which is receptive at this stage.
The disadvantage of each of these procedures is that the SI line, as a result of the high costs involved, can only be maintained on a small scale. Extensive field production of the hybrids using the SI line as female is impractical due to cost factors.